RESUMEN
Plants, unlike animals, possess a unique developmental plasticity, that allows them to adapt to changing environmental conditions. A fundamental aspect of this plasticity is their ability to undergo postembryonic de novo organogenesis. This requires the presence of regulators that trigger and mediate specific spatiotemporal changes in developmental programs. The phytohormone cytokinin has been known as a principal regulator of plant development for more than six decades. In de novo shoot organogenesis and in vitro shoot regeneration, cytokinins are the prime candidates for the signal that determines shoot identity. Both processes of de novo shoot apical meristem development are accompanied by changes in gene expression, cell fate reprogramming, and the switching-on of the shoot-specific homeodomain regulator, WUSCHEL. Current understanding about the role of cytokinins in the shoot regeneration will be discussed.
RESUMEN
While the pivotal role of linker histone H1 in shaping nucleosome organization is well established, its functional interplays with chromatin factors along the epigenome are just starting to emerge. Here we show that, in Arabidopsis, as in mammals, H1 occupies Polycomb Repressive Complex 2 (PRC2) target genes where it favors chromatin condensation and H3K27me3 deposition. We further show that, contrasting with its conserved function in PRC2 activation at genes, H1 selectively prevents H3K27me3 accumulation at telomeres and large pericentromeric interstitial telomeric repeat (ITR) domains by restricting DNA accessibility to Telomere Repeat Binding (TRB) proteins, a group of H1-related Myb factors mediating PRC2 cis recruitment. This study provides a mechanistic framework by which H1 avoids the formation of gigantic H3K27me3-rich domains at telomeric sequences and contributes to safeguard nucleus architecture.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Animales , Histonas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Cromatina , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/genética , Telómero/metabolismo , Mamíferos/metabolismoRESUMEN
Telomere repeat binding proteins (TRBs) belong to a family of proteins possessing a Myb-like domain which binds to telomeric repeats. Three members of this family (TRB1, TRB2, TRB3) from Arabidopsis thaliana have already been described as associated with terminal telomeric repeats (telomeres) or short interstitial telomeric repeats in gene promoters (telo-boxes). They are also known to interact with several protein complexes: telomerase, Polycomb repressive complex 2 (PRC2) E(z) subunits and the PEAT complex (PWOs-EPCRs-ARIDs-TRBs). Here we characterize two novel members of the TRB family (TRB4 and TRB5). Our wide phylogenetic analyses have shown that TRB proteins evolved in the plant kingdom after the transition to a terrestrial habitat in Streptophyta, and consequently TRBs diversified in seed plants. TRB4-5 share common TRB motifs while differing in several others and seem to have an earlier phylogenetic origin than TRB1-3. Their common Myb-like domains bind long arrays of telomeric repeats in vitro, and we have determined the minimal recognition motif of all TRBs as one telo-box. Our data indicate that despite the distinct localization patterns of TRB1-3 and TRB4-5 in situ, all members of TRB family mutually interact and also bind to telomerase/PRC2/PEAT complexes. Additionally, we have detected novel interactions between TRB4-5 and EMF2 and VRN2, which are Su(z)12 subunits of PRC2.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Telomerasa , Telomerasa/genética , Telomerasa/metabolismo , Filogenia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismo , Telómero/genética , Telómero/metabolismo , SueloRESUMEN
The proper development of male and female gametophytes is critical for successful sexual reproduction and requires a carefully regulated series of events orchestrated by a suite of various proteins. RUVBL1 and RUVBL2, plant orthologues of human Pontin and Reptin, respectively, belong to the evolutionarily highly conserved AAA+ family linked to a wide range of cellular processes. Previously, we found that RUVBL1 and RUVBL2A mutations are homozygous lethal in Arabidopsis. Here, we report that RUVBL1 and RUVBL2A play roles in reproductive development. We show that mutant plants produce embryo sacs with an abnormal structure or with various numbers of nuclei. Although pollen grains of heterozygous mutant plants exhibit reduced viability and reduced pollen tube growth in vitro, some of the ruvbl pollen tubes are capable of targeting ovules in vivo. Similarly, some ruvbl ovules retain the ability to attract wild-type pollen tubes but fail to develop further. The activity of the RUVBL1 and RUVBL2A promoters was observed in the embryo sac, pollen grains, and tapetum cells and, for RUVBL2A, also in developing ovules. In summary, we show that the RUVBL proteins are essential for the proper development of both male and particularly female gametophytes in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Humanos , Células Germinativas de las Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Polen , Reproducción , Tubo Polínico/genética , Tubo Polínico/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismoRESUMEN
The canonical DNA polymerases involved in the replication of the genome are unable to fully replicate the physical ends of linear chromosomes, called telomeres. Chromosomal termini thus become shortened in each cell cycle. The maintenance of telomeres requires telomerase-a specific RNA-dependent DNA polymerase enzyme complex that carries its own RNA template and adds telomeric repeats to the ends of chromosomes using a reverse transcription mechanism. Both core subunits of telomerase-its catalytic telomerase reverse transcriptase (TERT) subunit and telomerase RNA (TR) component-were identified in quick succession in Tetrahymena more than 30 years ago. Since then, both telomerase subunits have been described in various organisms including yeasts, mammals, birds, reptiles and fish. Despite the fact that telomerase activity in plants was described 25 years ago and the TERT subunit four years later, a genuine plant TR has only recently been identified by our group. In this review, we focus on the structure, composition and function of telomerases. In addition, we discuss the origin and phylogenetic divergence of this unique RNA-dependent DNA polymerase as a witness of early eukaryotic evolution. Specifically, we discuss the latest information regarding the recently discovered TR component in plants, its conservation and its structural features.
Asunto(s)
Evolución Biológica , Telomerasa/química , Telomerasa/fisiología , Animales , Eucariontes/clasificación , Eucariontes/genética , Eucariontes/metabolismo , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Filogenia , ARN/fisiología , Telómero/metabolismoRESUMEN
Characterization of protein-protein and protein-DNA interactions is critical to understand mechanisms governing the biology of cells. Here we describe optimized methods and their mutual combinations for this purpose: bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (Co-IP), yeast two-hybrid systems (Y2H), and chromatin immunoprecipitation (ChIP). These improved protocols detect trimeric complexes in which two proteins of interest interact indirectly via a protein sandwiched between them. They also allow isolation of low-abundance chromatin proteins and confirmation that proteins of interest are associated with specific DNA sequences, for example telomeric tracts. Here we describe these methods and their application to map interactions of several telomere- and telomerase-associated proteins and to purify a sufficient amount of chromatin from Arabidopsis thaliana for further investigations (e.g., next-generation sequencing, hybridization).
Asunto(s)
Arabidopsis/metabolismo , Proteínas de Unión al ADN/metabolismo , Imagen Óptica/métodos , Mapeo de Interacción de Proteínas/métodos , Telomerasa/metabolismo , Telómero/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Inmunoprecipitación de Cromatina/métodos , ADN de Plantas/metabolismo , Proteínas de Unión al ADN/aislamiento & purificación , Inmunoprecipitación/métodos , Unión Proteica , Técnicas del Sistema de Dos HíbridosRESUMEN
Telomerase maturation and recruitment to telomeres is regulated by several telomerase- and telomere-associated proteins. Among a number of proteins, human Pontin and Reptin play critical roles in telomerase biogenesis. Here we characterized plant orthologues of Pontin and Reptin, RuvBL1 and RuvBL2a, respectively, and show association of Arabidopsis thaliana RuvBL1 (AtRuvBL1) with the catalytic subunit of telomerase (AtTERT) in the nucleolus in vivo. In contrast to mammals, interactions between AtTERT and AtRuvBL proteins in A. thaliana are not direct and they are rather mediated by one of the Arabidopsis thaliana Telomere Repeat Binding (AtTRB) proteins. We further show that plant orthologue of dyskerin, named AtCBF5, is indirectly associated with AtTRB proteins but not with the AtRuvBL proteins in the plant nucleus/nucleolus, and interacts with the Protection of telomere 1 (AtPOT1a) in the nucleolus or cytoplasmic foci. Our genome-wide phylogenetic analyses identify orthologues in RuvBL protein family within the plant kingdom. Dysfunction of AtRuvBL genes in heterozygous T-DNA insertion A. thaliana mutants results in reduced telomerase activity and indicate the involvement of AtRuvBL in plant telomerase biogenesis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Telomerasa/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Dominio Catalítico , Nucléolo Celular/metabolismo , ADN Helicasas/metabolismo , Proteínas Nucleares , Filogenia , Proteínas de Unión al ARN/metabolismo , Telómero/metabolismo , Proteínas de Unión a Telómeros/metabolismoRESUMEN
Parallel research on multiple model organisms shows that while some principles of telomere biology are conserved among all eukaryotic kingdoms, we also find some deviations that reflect different evolutionary paths and life strategies, which may have diversified after the establishment of telomerase as a primary mechanism for telomere maintenance. Much more than animals, plants have to cope with environmental stressors, including genotoxic factors, due to their sessile lifestyle. This is, in principle, made possible by an increased capacity and efficiency of the molecular systems ensuring maintenance of genome stability, as well as a higher tolerance to genome instability. Furthermore, plant ontogenesis differs from that of animals in which tissue differentiation and telomerase silencing occur during early embryonic development, and the "telomere clock" in somatic cells may act as a preventive measure against carcinogenesis. This does not happen in plants, where growth and ontogenesis occur through the serial division of apical meristems consisting of a small group of stem cells that generate a linear series of cells, which differentiate into an array of cell types that make a shoot and root. Flowers, as generative plant organs, initiate from the shoot apical meristem in mature plants which is incompatible with the human-like developmental telomere shortening. In this review, we discuss differences between human and plant telomere biology and the implications for aging, genome stability, and cell and organism survival. In particular, we provide a comprehensive comparative overview of telomere proteins acting in humans and in Arabidopsis thaliana model plant, and discuss distinct epigenetic features of telomeric chromatin in these species.
Asunto(s)
Plantas/metabolismo , Telómero/metabolismo , Senescencia Celular/genética , Cromatina/metabolismo , Epigénesis Genética , Humanos , Telomerasa/metabolismoRESUMEN
In the published online version, the affiliations were mixed up. Corrected affiliation section is shown below. Also, the update has also been reflected in the author group section above.
RESUMEN
The life cycle of telomerase involves dynamic and complex interactions between proteins within multiple macromolecular networks. Elucidation of these associations is a key to understanding the regulation of telomerase under diverse physiological and pathological conditions from telomerase biogenesis, through telomere recruitment and elongation, to its non-canonical activities outside of telomeres. We used tandem affinity purification coupled to mass spectrometry to build an interactome of the telomerase catalytic subunit AtTERT, using Arabidopsis thaliana suspension cultures. We then examined interactions occurring at the AtTERT N-terminus, which is thought to fold into a discrete domain connected to the rest of the molecule via a flexible linker. Bioinformatic analyses revealed that interaction partners of AtTERT have a range of molecular functions, a subset of which is specific to the network around its N-terminus. A significant number of proteins co-purifying with the N-terminal constructs have been implicated in cell cycle and developmental processes, as would be expected of bona fide regulatory interactions and we have confirmed experimentally the direct nature of selected interactions. To examine AtTERT protein-protein interactions from another perspective, we also analysed AtTERT interdomain contacts to test potential dimerization of AtTERT. In total, our results provide an insight into the composition and architecture of the plant telomerase complex and this will aid in delineating molecular mechanisms of telomerase functions.
Asunto(s)
Proteínas de Arabidopsis/aislamiento & purificación , Arabidopsis/enzimología , Telomerasa/aislamiento & purificación , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/enzimología , Células Cultivadas , Cromatografía de Afinidad , Expresión Génica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Multimerización de Proteína , Espectrometría de Masas en Tándem , Telomerasa/genética , Telomerasa/metabolismoRESUMEN
Telomeres, as physical ends of linear chromosomes, are targets of a number of specific proteins, including primarily telomerase reverse transcriptase. Access of proteins to the telomere may be affected by a number of diverse factors, e.g., protein interaction partners, local DNA or chromatin structures, subcellular localization/trafficking, or simply protein modification. Knowledge of composition of the functional nucleoprotein complex of plant telomeres is only fragmentary. Moreover, the plant telomeric repeat binding proteins that were characterized recently appear to also be involved in non-telomeric processes, e.g., ribosome biogenesis. This interesting finding was not totally unexpected since non-telomeric functions of yeast or animal telomeric proteins, as well as of telomerase subunits, have been reported for almost a decade. Here we summarize known facts about the architecture of plant telomeres and compare them with the well-described composition of telomeres in other organisms.
RESUMEN
Recently we characterised TRB1, a protein from a single-myb-histone family, as a structural and functional component of telomeres in Arabidopsis thaliana. TRB proteins, besides their ability to bind specifically to telomeric DNA using their N-terminally positioned myb-like domain of the same type as in human shelterin proteins TRF1 or TRF2, also possess a histone-like domain which is involved in protein-protein interactions e.g., with POT1b. Here we set out to investigate the genome-wide localization pattern of TRB1 to reveal its preferential sites of binding to chromatin in vivo and its potential functional roles in the genome-wide context. Our results demonstrate that TRB1 is preferentially associated with promoter regions of genes involved in ribosome biogenesis, in addition to its roles at telomeres. This preference coincides with the frequent occurrence of telobox motifs in the upstream regions of genes in this category, but it is not restricted to the presence of a telobox. We conclude that TRB1 shows a specific genome-wide distribution pattern which suggests its role in regulation of genes involved in biogenesis of the translational machinery, in addition to its preferential telomeric localization.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Secuencia de Bases , Biología Computacional , Biblioteca de Genes , Histonas/metabolismo , Datos de Secuencia Molecular , Motivos de Nucleótidos , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , Unión Proteica , Biosíntesis de Proteínas , Ribosomas/genética , Análisis de Secuencia de ADN , Proteínas de Unión a Telómeros/genéticaRESUMEN
Although telomere-binding proteins constitute an essential part of telomeres, in vivo data indicating the existence of a structure similar to mammalian shelterin complex in plants are limited. Partial characterization of a number of candidate proteins has not identified true components of plant shelterin or elucidated their functional mechanisms. Telomere repeat binding (TRB) proteins from Arabidopsis thaliana bind plant telomeric repeats through a Myb domain of the telobox type in vitro, and have been shown to interact with POT1b (Protection of telomeres 1). Here we demonstrate co-localization of TRB1 protein with telomeres in situ using fluorescence microscopy, as well as in vivo interaction using chromatin immunoprecipitation. Classification of the TRB1 protein as a component of plant telomeres is further confirmed by the observation of shortening of telomeres in knockout mutants of the trb1 gene. Moreover, TRB proteins physically interact with plant telomerase catalytic subunits. These findings integrate TRB proteins into the telomeric interactome of A. thaliana.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Telomerasa/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Arabidopsis/genéticaRESUMEN
Chromosome stability is conditioned by functional chromatin structure of chromosome ends - telomeres. Organisation and regulation of telomere maintenance represent a complex process whose details still remain enigmatic, especially in plants. Several telomere-binding or telomere-associated proteins and distinct epigenetic marks have been shown to influence telomere length and telomerase activity. HMGB proteins play important role in dynamic changes of chromatin structure and are involved in regulation of cellular processes of key importance, such as replication, transcription, recombination and DNA-repair. HMGB proteins in plants are more diversified than in other eukaryotes. Here, we summarise the roles of plant HMGB proteins in regulation of chromatin structure and dynamics and report on the newly identified role of AtHMGB1 protein in the regulation of plant telomere length. Astonishingly, contrary to mice mHMGB1 homologue, AtHMGB1 does not affect telomerase activity and AtHMGB1 loss or overexpression does not cause any obvious changes in chromatin architecture.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cromatina/metabolismo , Proteínas HMGB/metabolismo , Proteína HMGB1/metabolismo , Telómero/metabolismo , Cromatina/química , EpigenómicaRESUMEN
Telobox is a Myb-related DNA-binding domain which is present in a number of yeast, plant and animal proteins. Its capacity to bind preferentially double-stranded telomeric DNA has been used in numerous studies to search for candidate telomeric proteins in various organisms, including plants. Here we provide an overview of these studies with a special emphasis on plants, where a specific subfamily of the proteins possessing the N-terminally positioned telobox is present in addition to more common C-terminal telobox proteins. We further demonstrate the presence of a telobox protein (CpTBP1) in Cestrum parqui, a plant lacking typical telomeres and telomerase. The protein shows nuclear localisation and association with chromatin. The role of this protein in ancestral and current telomere structure is discussed in the evolutionary context. Altogether, the present overview shows the importance of the telobox domain in a search for candidate telomere proteins but at the same time warns against oversimplified identification of any telobox protein with telomere structure without appropriate evidence of its telomeric localisation and function.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Secuencia de Aminoácidos , Cestrum/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/genética , Datos de Secuencia Molecular , Filogenia , Proteínas de Plantas/química , Proteínas de Unión a Telómeros/genéticaRESUMEN
Proteins that bind telomeric DNA modulate the structure of chromosome ends and control telomere function and maintenance. It has been shown that AtTRB (Arabidopsis thaliana telomere-repeat-binding factor) proteins from the SMH (single-Myb-histone) family selectively bind double-stranded telomeric DNA and interact with the telomeric protein AtPOT1b (A. thaliana protection of telomeres 1b), which is involved in telomere capping. In the present study, we performed the first quantitative DNA-binding study of this plant-specific family of proteins. Interactions of full-length proteins AtTRB1 and AtTRB3 with telomeric DNA were analysed by electrophoretic mobility-shift assay, fluorescence anisotropy and surface plasmon resonance to reveal their binding stoichiometry and kinetics. Kinetic analyses at different salt conditions enabled us to estimate the electrostatic component of binding and explain different affinities of the two proteins to telomeric DNA. On the basis of available data, a putative model explaining the binding stoichiometry and the protein arrangement on telomeric DNA is presented.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Histonas/metabolismo , Telómero/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ensayo de Cambio de Movilidad Electroforética , Polarización de Fluorescencia , Histonas/genética , Cinética , Modelos Biológicos , Unión Proteica/genética , Resonancia por Plasmón de Superficie , Telómero/genéticaRESUMEN
Telomeres are nucleoprotein structures ensuring the stability of eukaryotic chromosome ends. Two protein families, TRFL (TFL-Like) and SMH (Single-Myb-Histone), containing a specific telobox motif in their Myb domain, have been identified as potential candidates involved in a functional nucleoprotein structure analogous to human "shelterin" at plant telomeres. We analyze the DNA-protein interaction of the full-length and truncated variants of AtTRB1, a SMH-family member with a typical structure: N-terminal Myb domain, central H1/5 domain and C-terminal coiled-coil. We show that preferential interaction of AtTRB1 with double-stranded telomeric DNA is mediated by the Myb domain, while the H1/5 domain is involved in non-specific DNA-protein interaction and in the multimerization of AtTRB1.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Unión al ADN/metabolismo , Telómero/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Clonación Molecular , ADN de Plantas/metabolismo , Proteínas de Unión al ADN/genética , Electroforesis en Gel de Poliacrilamida , Histonas/metabolismo , Humanos , Proteínas Oncogénicas v-myb/metabolismo , Unión ProteicaRESUMEN
We previously searched for interactions between plant telomere-binding proteins and found that AtTRB1, from the single-myb-histone (Smh) family, interacts with the Arabidopsis POT1-like-protein, AtPOT1b, involved in telomere capping. Here we identify domains responsible for that interaction. We also map domains in AtTRB1 responsible for interactions with other Smh-family-members. Our results show that the N-terminal OB-fold-domain of AtPOT1b mediates the interaction with AtTRB1. This domain is characteristic for POT1- proteins and is involved with binding the G-rich-strand of telomeric DNA. AtPOT1b also interacts with AtTRB2 and AtTRB3. The central histone-globular-domain of AtTRB1 is involved with binding to AtTRB2 and 3, as well as to AtPOT1b. AtTRB1-heterodimers with other Smh-family-members are more stable than AtTRB1-homodimers. Our results reveal interaction networks of plant telomeres.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas de Unión a Telómeros/metabolismo , Secuencia de Aminoácidos , Proteínas de Arabidopsis/genética , Clonación Molecular , Dimerización , Humanos , Datos de Secuencia Molecular , Proteínas de Unión a Telómeros/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Telomeres in many eukaryotes are maintained by telomerase in whose absence telomere shortening occurs. However, telomerase-deficient Arabidopsis thaliana mutants (Attert (-/-)) show extremely low rates of telomere shortening per plant generation (250-500 bp), which does not correspond to the expected outcome of replicative telomere shortening resulting from ca. 1,000 meristem cell divisions per seed-to-seed generation. To investigate the influence of the number of cell divisions per seed-to-seed generation, Attert (-/-) mutant plants were propagated from seeds coming either from the lower-most or the upper-most siliques (L- and U-plants) and the length of their telomeres were followed over several generations. The rate of telomere shortening was faster in U-plants, than in L-plants, as would be expected from their higher number of cell divisions per generation. However, this trend was observed only in telomeres whose initial length is relatively high and the differences decreased with progressive general telomere shortening over generations. But in generation 4, the L-plants frequently show a net telomere elongation, while the U-plants fail to do so. We propose that this is due to the activation of alternative telomere lengthening (ALT), a process which is activated in early embryonic development in both U- and L-plants, but is overridden in U-plants due to their higher number of cell divisions per generation. These data demonstrate what so far has only been speculated, that in the absence of telomerase, the number of cell divisions within one generation influences the control of telomere lengths. These results also reveal a fast and efficient activation of ALT mechanism(s) in response to the loss of telomerase activity and imply that ALT is probably involved also in normal plant development.
Asunto(s)
Arabidopsis/genética , Mutación , Telomerasa/genética , Telómero/genética , Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , División Celular/genética , División Celular/fisiología , Cromosomas de las Plantas/genética , Semillas/enzimología , Semillas/genética , Semillas/crecimiento & desarrollo , Telomerasa/metabolismo , Telómero/metabolismoRESUMEN
Telomere-binding proteins participate in forming a functional nucleoprotein structure at chromosome ends. Using a genomic approach, two Arabidopsis thaliana genes coding for candidate Myb-like telomere binding proteins were cloned and expressed in E. coli. Both proteins, termed AtTBP2 (accession Nos. T46051 (protein database) and GI:638639 (nucleotide database); 295 amino acids, 32 kDa, pI 9.53) and AtTBP3 (BAB08466, GI:9757879; 299 amino acids, 33 kDa, pI 9.88), contain a single Myb-like DNA-binding domain at the N-terminus, and a histone H1/H5-like DNA-binding domain in the middle of the protein sequence. Both proteins are expressed in various A. thaliana tissues. Using the two-hybrid system interaction between the proteins AtTBP2 and AtTBP3 and self interactions of each of the proteins were detected. Gel-retardation assays revealed that each of the two proteins is able to bind the G-rich strand and double-stranded DNA of plant telomeric sequence with an affinity proportional to a number of telomeric repeats. Substrates bearing a non-telomeric DNA sequence positioned between two telomeric repeats were bound with an efficiency depending on the length of interrupting sequence. The ability to bind variant telomere sequences decreased with sequence divergence from the A. thaliana telomeric DNA. None of the proteins alone or their mixture affects telomerase activity in vitro. Correspondingly, no interaction was observed between any of two proteins and the Arabidopsis telomerase reverse transcriptase catalytic subunit TERT (accession No. AF172097) using two-hybrid assay.